Hepatitis C virus (HCV) is a major causative agent of post-transfusion and community-acquired non-A, non-B hepatitis world-wide (Kuo, G. et al., Science 244:362-364, 1989; Choo O. L. et al., Science 244:359-362, 1989; Alter H. J. et al., N. Engl. J. Med. 321:1494-1500, 1989; Kato N. et al., Proc. Natl. Acad. Sci. USA 87: 9524-9528, 1990). The majority of HCV infected individuals develop chronic hepatitis which progresses eventually to liver cirrhosis and hepatocellular carcinoma (Tong M. J. et al., N. Engl. J. Med. 332:1463-1466, 1995). Currently, no effective vaccine to prevent HCV infection or treatment for chronic HCV infection exists (Lemon, S. M. and Thomas, D. L., New Engl. J. Med. 336:177-203, 1997; Hoofuagle, J. and DiBisceclie, New Engl. J. Med. 336:347-356, 1997). Development of an effective vaccine and/or treatment has been hampered by the inability to propagate HCV efficiently in cultured cells and the lack of a small animal model.
HCV is a member of the flavivirus family (; Francki R. I. B. et al., Arch. Virol., Suppl. 2.223-233, 1991). The HCV virion contains a positive-strand RNA genome of 9.5 kilobases (kb) including a highly conserved 5xe2x80x2 noncoding region followed by a long open reading frame of 9030 to 9099 nucleotides (nt) that is translated into a single polyprotein of about 3,010 to 3,030 amino acids (Matsuura Y. and Miyamura T., Seminars in Virol. 4:297-304, 1993; Hijikata M. et al., Proc. Natl. Acad. Sci. USA 88:5547-5551, 1991). Initiation of translation occurs by a mechanism of internal ribosomal entry requiring the 5xe2x80x2 untranslated region (UTR) and a short stretch of HCV coding sequences (Reynolds J. E. et al, EMBO J. 14:6010-6020, 1995). Processing of the polyprotein occurs by a combination of host and viral proteases to produce at least ten putative viral structural and nonstructural (NS) proteins. The HCV structural proteins comprise the nucleocapsid or core protein (C) and the two putative virion envelope glycoproteins E1 and E2 (Miyamura T. and Matsuura Y., Trends Microbiol. 1(6):229-231, 1993). The cleavage of structural proteins from the polyprotein is catalyzed by a host signal peptidase (Hijikata M. et al., Proc. Natl. Acad. Sci. USA 88:5547-5551, 1991; Lin C. et al., J. Virol. 68(8):5063-5073, 1994), whereas polyprotein cleavage in the nonstructural region requires the presence of HCV-encoded proteinases encoded by the nonstructural region (Grakoui A. et al., Proc. Natl. Acad. Sci. USA 90:10583-10587, 1993).
Although the viral genomic organization has been characterized in detail, morphologic analysis of hepatitis C virus has been hampered by low levels of HCV particles in infected patients and the inability to propagate efficiently the virus in cultured cells. The levels of the viral particles present in infected patient plasma and/or liver tissues are very low, making it difficult to visualize the virus. By analogy to other members of the Flaviviridae, the HCV genomic organization suggests a virus consisting of a nucleocapsid comprising a viral genome and core protein coated by a lipid envelope containing the envelope glycoproteins E1 and E2. Studies of HCV infection in chimpanzees, a reliable animal model for hepatitis C, have provided evidence that HCV is inactivated by chloroform, indicating that it contains essential lipids and therefore is probably enveloped (Feinstone, S. M. et al., Infect. Immun. 41:816-821, 1983). Filtration studies have estimated the virion particle size to be about 30-60 nm in diameter (He et al., J. Infect. Dis. 156:636-640, 1987).
Recombinant HCV proteins have been produced using various expression systems, but no virus-like particles have been generated in these systems (Grakoui A. et al., J. Virol. 67:1385-1395, 1993; Hijikata, M. et al., Proc. Natl. Acad. Sci. USA 88:5547-5551, 1991; LauFord, B. et al., Virol. 197:225-235, 1993; Miyamura, T. and Matsuura, Y., Trends Microbiol. 1:229-231, 1993). Production of recombinant HCV proteins suggests that some of the HCV proteins specifically interact. For example, previous results suggest that the HCV core protein interacts with the E1 envelope protein but not with the E2 envelope protein (Lo S.-Y. et al., J. Virol. 70(6): 5177-5182, 1996). Recombinant HCV polypeptides produced in vitro have been disclosed in PCT application WO 9604301, PCT application WO 9533053, PCT application WO 9102820 and U.S. Pat. No. 5,372,928.
Virus-like particles have been synthesized for viruses of various families other than Flaviviridae or Pestiviridae using a baculovirus-insect cell expression system (Gheysen D. et al., Cell 59:103-112, 1989; Kirnbauer R. et al., Proc. Natl. Acad. Sci. USA 89:12180-12184; 1992; Zeng C. O.-Y. et al., J. Virol. 70:2736-2742, 1996). The baculovirus-insect cell expression of viral proteins is advantageous because the eukaryotic insect cells can carry out a number of co- or post-translational modifications such as fatty acid acetylation and glycosylation, similar to mammalian cells (Luckow, V. A. and Summers, M. D., Virol. 167:56, 1988). Moreover, the baculovirus expression system allows higher levels of heterologous protein synthesis than generally is possible in many mammalian cell expression systems (Luckow, V. A. and Summers, M. D., Viral. 167:56, 1988).
The present invention differs from the prior art because it utilizes a recombinant construct that contains nucleic acid that includes part of the 5xe2x80x2 UTR, coding sequences for HCV structural proteins including p7, and produces virus-like particles when the construct is expressed in insect cells. These virus-like particles of an enveloped RNA virus are generated without other components required for viral replication and are assembled intracellularly in vitro. These virus-like particles are effective immunogens for generating HCV-specific antibodies and thus are important for development of an effective HCV vaccine.
According to one aspect of the invention, there is provided a method of producing virus-like particles in vitro comprising the steps of providing a vector comprising an expression system capable of producing proteins in insect cells, cloning a cDNA that codes for structural proteins of an enveloped RNA virus into the vector such that the cDNA is capable of being expressed in transfected or infected insect cells, transfecting or infecting insect cells with the vector containing the cloned cDNA that codes for the structural proteins of an enveloped RNA virus, maintaining the transfected or infected insect cells in culture for sufficient time to allow expression of the cDNA to produce the structural proteins of an enveloped RNA virus, and allowing the structural proteins to form intracellular virus-like particles. In one embodiment, the method further comprises the step of purifying the intracellular virus-like particles from the cultured cells. In a preferred embodiment, the purifying step comprises lysing the cells to produce a lysate and subjecting the lysate to gradient centrifugation. In another embodiment, the purifying step comprises lysing the cells to produce a lysate and subjecting the lysate to immunoadsorption using an immunoreagent that specifically recognizes a viral protein contained within the virus-like particles. In one embodiment, the method further comprises the step of generating an immune response in a mammal by introducing an effective amount of purified virus-like particles into the mammal in a pharmaceutically acceptable carrier. In a preferred embodiment, the immunizing step is performed in a mammal selected from the group consisting of a mouse, rat, rabbit, goat, sheep, horse and human. In one embodiment of the method, the cloned cDNA is produced from an enveloped RNA virus that is a member of the Sindbis-like superfamily or a member of the Flavivirus-like superfamily. Preferrably, the cloned cDNA is produced from a member of the group consisting of Togaviridae, Bromovirus, Cucumovirus, Tobavirus, Ilarvirus, Tobravirus, Potexvirus, Flaviviridae, and Pestivirus. In one embodiment of the method, the cloning step comprises cloning a cDNA comprising a 5xe2x80x2 untranslated region and sequences coding for hepatitis C virus (HCV) core protein, HCV envelope 1 (E1) protein, HCV envelope 2 (E2) protein and p7 protein, such that the cDNA is capable of being expressed in transfected or infected insect cells, and the maintaining step comprises maintaining the transfected or infected insect cells in culture for about 72 hr to 120 hr, to allow expression of the cDNA to produce HCV structural proteins and allow the HCV structural proteins to form intracellular HCV-like particles. The cloned cDNA may also include sequence that codes for a few amino acids of non-structural protein NS2. One embodiment of the invention is HCV-like particles produced according to this method. In one embodiment, the HCV-like particles further comprise a portion of HCV RNA transcript. The HCV-like particles are about 40 nm to about 60 nm in diameter and have a density of about 1.14 g/cm3 to about 1.18 g/cm3. Another embodiment of the invention is a vaccine comprising the HCV-like particles in a pharmaceutically acceptable carrier. One more embodiment is a therapeutic agent comprising HCV-like particles in a pharmaceutically acceptable carrier. Another embodiment is antibodies produced by immunizing an animal with HCV-like particles, and the antibodies can be monoclonal antibodies. Another embodiment of the invention is a diagnostic kit for detecting HCV infection in an individual comprising HCV-like particles and a, means for detecting antibodies that bind to the HCV-particles.
According to a second aspect of the invention, there is provided a recombinant construct comprising a vector comprising an expression system capable of producing proteins in insect cells in vitro and a DNA complementary (cDNA) to hepatitis C virus (HCV) RNA, wherein the cDNA comprises a 5xe2x80x2 untranslated region and sequences coding for HCV core protein, HCV envelope 1 (E1) protein, HCV envelope 2 (E2) protein and p7 protein, such that the cDNA is capable of being expressed in insect cells transfected or infected with the recombinant construct. The recombinant construct may also include sequence coding for at least a part of HCV NS2 protein. One embodiment of this aspect of the invention is insect cells transfected or infected with the recombinant construct.
According to a third aspect of the invention, there are provided recombinant HCV proteins comprising HCV core protein, HCV envelope 1 (E1) protein, HCV envelope 2 (E2) protein and p7 protein that assemble into intracellular HCV-like particles in insect cells.
It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. The accompanying figures, which are incorporated in and constitute a part of the specification, illustrate various embodiments of the invention and, together with the description, serve to explain the principles of the invention.